This proposal tests several related hypotheses concerning mechanisms of neuronal injury and death relevant to the pathogenesis of Alzheimer's disease (AD). Studies performed in rat hippocampal cell cultures will test: (1) The hypothesis that glutamate and Abeta impair function of the plasma membrane Na+/K+ ATPase and Ca2+ ATPase by a free radical-mediated mechanism involving inositol phospholipid hydrolysis. This will be accomplished using sensitive ATPase activity assays, and fluorescence imaging technologies to characterize temporal changes in free radical levels [Na+]i, and [Ca2+]i. (2) The hypothesis that neurotrophic factors protect neurons against Abeta and glutamate toxicities by increasing expression of antioxidant enzymes, suppressing free radical accumulation, and preventing impairment of Na+/K+ and Ca2+ ATPase activities. (3) The hypothesis that MAP kinases mediate the effects of neurotrophic factors on antioxidant enzyme levels, free radical accumulation, loss of ion homeostasis and cell death. This will be accomplished using manipulations of MAP kinase activity using tyrosine kinase inhibitors and activators, and antisense oligodeoxynucleotides to deplete MAP kinase levels. (4) The hypothesis that concomitant activation of neurodegenerative and neuroprotective signaling pathways can result in alterations in localization and phosphorylation of tau similar to those seen in neurofibrillary tangles - neurons will be induced to degenerate under conditions where MAP kinases are activated. The final aim will employ synaptic membrane preparations from vulnerable and non-vulnerable regions of AD control brains. The hypothesis that ion-motive ATPases are impaired in brain regions that are particularly vulnerable in AD will be tested and the effects of Abeta on ATPase activities will be determined. This research will employ a battery of technologies including: hippocampal cell culture; synaptic membrane preparations; ATPase activity assays; measurements of free radical levels and intracellular Na+ and Ca2+ levels using fluorescent indicator dyes; antisense techniques to suppress expression of MAP kinases; Western blot analysis; chromatographic analyses of inositol phospholipid metabolites. The data generated will provide fundamental information concerning the roles of free radicals in the impairment of ion homeostatic mechanisms by glutamate and Abeta, and the mechanism whereby neurotrophic factors increase resistance of neurons to excitotoxicity and Abeta toxicity. The relevance of such mechanisms to the pathogenesis of AD will be established in studies of ion-motive ATPases in synaptic membranes from postmortem AD and control human brains. This research is likely to identify cellular targets to aim at in the design of prophylactic and therapeutic interventions in AD and related neurodegenerative disorders.